The use of systemic chemotherapy and radiation therapy for treatment of malignancies has failed to effectively alter the progress of malignant tumors of the central nervous system. The fatal outcome resulting from malignancies such as prostatic and mammary malignancies is often due to inability of current chemotherapy to effectively reach malignant growths in the central nervous system.
The use of cytotoxic products in the treatment of cancer is well known. The difficulties associated with such treatment are also well known. Of these difficulties, the lack of cancer-specific cytotoxicity has received considerable attention, albeit resolution of these difficulties has met with marginal success. Cytotoxic products kill normal cells as well as cancer cells. Such non-specificity results in a number of undesirable side effects for patients undergoing cancer chemotherapy with cytotoxic products, including nausea, vomiting, diarrhea, hemorrhagic gastroenteritis, and hepatic and renal damage. Due to normal cell toxicity, the therapeutic dosage of cytotoxic products has been limited such that cancerous cells are not killed to a sufficient level that subsequently prevents or delays new cancerous growth.
Current approaches to cancer chemotherapy and other immunological therapies focus on the use of cell-specific antibodies bonded to toxins in order to kill specific populations of cancer cells. Immunotoxins (protein toxins chemically linked to tumor-specific monoclonal antibodies or other ligands) offer potential advantages over more conventional forms of treatment by having higher tumor specificity. Ideally, immunotoxins should discriminate to a high degree between target and non-target cells. The critical point, then, is the development of immunotoxins that are highly toxic for specific populations of cells.
Monoclonal antibodies linked to toxic proteins (immunotoxins) can selectively kill some tumor cells in vitro and in vivo. However, reagents that combine the full potency of the native toxins with the high degree of cell-type selectivity of monoclonal antibodies have not previously been designed.
Immunotoxins may be particularly efficacious for the treatment of neoplastic disease confined to compartments such as the peritoneum or intrathecal space. Direct delivery into the compartment avoids complications associated with systemic delivery and produces relatively high local concentrations, thereby achieving greater therapeutic effects. The cerebrospinal fluid compartment may be amenable to this type of compartmentalized immunotoxin treatment. Zovickian and Youle, J Neurosurg, in press, examined the therapeutic effect of a monoclonal antibody-ricin immunotoxin delivered directly into the CSF compartment in a guinea pig model of leptomenigeal neoplasia.
To investigate the efficacy of intrathecal immunotoxin therapy for tumors of the CSF compartment, a simple model of leptomeningeal neoplasia was developed with direct inoculation of L.sub.2 C leukemia cells into the cisterna magna of Strain 2 guinea pigs. Animals were anesthetized with intraperitoneal ketamine (30 to 50 mg/kg). Viable L.sub.2 C cells suspended in 100 .mu.l PBS were slowly injected percutaneously via a No. 25 needle into the cisterna magna. Injections were performed only after CSF was clearly visualized in the hub of the needle.
Injection of 1.times.10.sup.5 viable L.sub.2 C cells (10,000 times the lethal dose) into the cisterna magna was performed. Twenty-four hours later, the animals were reanesthetized with ketamine and were injected with a single dose of either M6-ricin immunotoxin, control MOPC 21-ricin immunotoxin, M6 monoclonal antibody, or PBS, again via percutaneous puncture of the cisterna magna with a No. 25 needle. All agents were injected in a final volume of 100 .mu.l PBS. Length of survival was recorded as the number of days following tumor cell inoculation until death.
Percutaneous inoculation of 10 to 10.sup.5 L.sub.2 C cells into the cisterna magna of Strain 2 guinea pigs resulted in clinical and histological evidence of CNS disease. Clinically, animals variably exhibited irritability, paresis, head-tilting, ataxia, and seizures, with rapid progression to a terminal stage of prostration and death. Some animals developed palpable subcutaneous tumor nodules in the neck at the site of percutaneous tumor cell injection.
Histological evaluation of the brains removed from four animals at the terminal stage of disease showed extensive leptomeningeal and ependymal leukemic infiltration. Densely packed tumor cells layered the surfaces of the brains and extended perivascularly along Virchow-Robin spaces. There was diffuse invasion of the ventricular system by tumor cells. In some areas, the pia-glial membranes were disrupted, and nodules of neoplastic cells penetrated the brain parenchyma. Cytological evaluation of CSF aspirated from the cisterna magna of an animal at a terminal stage of disease revealed numerous lymphoblasts.
Animals treated with M6-ricin immunotoxin survived significantly longer than did control animals that received either PBS, M6 monoclonal antibody, or non-specific MOPC 21-ricin immunotoxin. The median survival time for control animals that received either PBS (six animals) or nonspecific MOPC 21-ricin immunotoxin (five animals) was 15 days, and for the eight control animals that received M6 monoclonal antibody was 15.5 days. In contrast, the 13 animals treated with M6-ricin immunotoxin survived from 16 to 27 days, with a median survival time of 20 days. This 5-day extension of median survival time for M6-ricin immunotoxin-treated animals is highly significant (p&lt;0.005) when compared to any of the control groups, and corresponds to a median 2- to 3-log (99% to 99.9%) tumor cell kill.
The therapeutic benefit observed may well represent a "worst case scenario." Owing to the guinea pig's small size, percutaneous cisterna magna puncture is necessarily a crude method for tumor cell inoculation and immunotoxin delivery. A certain amount of CSF leakage of injected tumor cells and immunotoxin undoubtedly occurred. Furthermore, it is likely that an occasional injection (or portion thereof) of tumor cells and/or immunotoxin was delivered subdurally or epidurally and not into the CSF compartment. Small numbers of tumor cells were undoubtedly deposited along the needle track at the time of intracisternal tumor cell injection. Palpable tumor nodules were occasionally observed at the site of tumor cell inoculation. Since as few as 10 cells deposited intradermally cause death from peripheral leukemia, those animals with even very small extrathecal tumor deposits would eventually die from peripheral leukemia not accessible to intrathecally administered immunotoxin, even if the intrathecal immunotoxin eradicated the central disease. The fact that 50% of the animals that received an intracisternal injection of tumor cells had hematological evidence of peripheral leukemia at the terminal stage supports this possibility. Therefore, a median 2- to 3-log kill may underestimate the cell kill achieved in the CSF compartment. Those animals (16%) that survived longer than 44 days, or the occasional animal that survived long-term (corresponding to a 5-log or greater tumor cell kill) may more accurately reflect the actual therapeutic effect in the CSF compartment in the absence of peripheral disease.
Protein toxins used in the constructions of immunotoxins have an A and a B subunit. The A subunit catalyzes the inactivation of protein synthesis, resulting ultimately in cell death. The B subunit has two functions: it is responsible for toxin binding to the cell surface, and it facilitates the translocation of the A chain across the membrane and into the cytosol, where the A chain acts to kill cells.
Previously, two general types of immunotoxins have been used. Immunotoxins made with the complete toxin molecule, both A and B chains, have the complication of non- specific killing mediated by the toxin B chain binding site. This can be avoided by eliminating the B chain and linking only the A chain to the antibody. However, A chain immunotoxins, although more specific, are much less toxic to tumor cells. The B chain, in addition to having a binding function, also has an entry function, which facilitates the translocation of the A chain across the membrane and into the cytosol. Since A-chain immunotoxins lack the entry function of the B chain, they are less toxic than their intact toxin counterparts containing the complete B chain. An ideal toxin for immunotoxin construction would contain the A chain enzymatic function and the B chain translocation function, but not the B chain binding function.
Two heretofore inseparable activities on one polypeptide chain of diphtheria toxin and ricin account for the failure to construct optimal reagents. The B-chains facilitate entry of the A-chain to the cytosol, allowing immunotoxins to kill target cells efficiently and bind to receptors present on most cells, imparting immunotoxins with a great degree of non-target-cell toxicity.
Some toxins have been modified to produce a suitable immunotoxin. The two best known are ricin and diphtheria toxin. Antibodies which bind cell surface antigens have been linked to diphtheria toxin and ricin, forming a new pharmacologic class of cell type-specific toxins. Ricin and diphtheria toxin are 60,000 to 65,000 dalton proteins with two subunits: the A-chain inhibits protein synthesis when in the cytosol, and the B-chain binds cell surface receptors and facilitates passage of the A subunit into the cytosol. Two types of antibody-toxin conjugates (immunotoxins) have been shown to kill antigen-positive cells in vitro. Immunotoxins made by binding only the toxin A subunit to an antibody have little non-target cell toxicity, but are often only minimally toxic to antigen-positive cells. Another type of immunotoxin is made by linking the whole toxin, A and B subunits, to the antibody and blocking the binding of the B subunit to prevent toxicity to non-target cells. For ricin, the non-target cell binding and killing can be blocked by adding lactose to the culture media or by steric restraint imposed by linking ricin to the antibody. Intact ricin immunotoxins may have only 30- to 100-fold selectivity between antigen-positive and negative cells, but they are highly toxic, and the best reagents can specifically kill a great many target cells.
Intact ricin and ricin A-chain immunotoxins have been found to deplete allogenic bone marrow of T cells, which can cause graft-versus-host diseases (GVHD), or to deplete autologous marros of tumor cells.
Diphtheria toxin is composed of two disulfide-linked subunits: the 21,000 dalton A-chain inhibits protein synthesis by catalyzing the ADP-riboxylation of elongation factor 2, and the 37,000-dalton B-chain binds cell surface receptors and facilitate transport of the A-chain to the cytosol. A single molecule of either a diphtheria toxin A-chain or a ricin A-chain in the cytosol is sufficient to kill a cell. The combination of these three activities, binding, translocation, and catalysis, produces the extreme potency of these proteins. The cell surface-binding domain and the phosphate-binding site are located within the carboxyl-terminal 8-kDa cyanogen bromide peptide of the B-chain. Close to the C-terminus region of the B-chain are several hydrophobic domains that can insert into membranes at low pH and appear to be important for diphtheria toxin entry.
Antibodies directed against cell surface antigens have been linked to intact diphtheria toxin or its A subunit to selectively kill antigen-bearing target cells. Antibody-toxin (immunotoxins) or ligand toxin conjugates containing only the diphtheria A-chain have relatively low cytotoxic activity. Intact diphtheria toxin conjugates can be very potent, but can also have greater toxicity to normal cells. Since the B-chain appears to facilitate entry of the A-chain to the cytosol, it is possible that its presence in whole toxin conjugates renders them more potent, although less specific. Efforts have been made to construct more potent and specific immunotoxins by separating the toxin B-chain domains involved in cell binding from the domains involved in A-chain entry.
Target cell toxicity of immunotoxins can be increased by including the toxin B-chain in the antibody-toxin complex or by adding it separately. To achieve maximal in vitro target-cell selectivity with immunotoxins containing intact ricin, lactose must be added to the medium to block non-target-cell binding and toxicity of the immunotoxin via the ricin B-chain. This approach is feasible in those clinical settings, such as bone marrow transplantation, where the target cell population can be incubated in vitro in the presence of lactose. Without blockage of the B-chain binding domain, however, whole toxin conjugates have a high degree of non-target-cell toxicity, thereby limiting their usefulness in vivo.
Construction of reagents that combine the potency of intact toxin conjugates with the cell-type selectivity of toxin A-chain conjugates may be possible if the binding site on the toxin B-chain could be irreversibly blocked. Covalent and noncovalent chemical modifications that block the binding activity of ricin intracellularly also block its entry function, suggesting that the binding and translocation functions may be inseparable.
Previously, domain deletion was unsuccessfully used in an attempt to separate the translocation and the binding functions of diphtheria toxin B-chain. Immunotoxins made with the A-chain, intact diphtheria toxin, and a cloned fragment of diphtheria toxin (MspSA) that lacks the C-terminal 17-kDa region of the B subunit were compared. The intact diphtheria conjugate was 100 times more toxic than the MspSA conjugate was, which, in turn, was 100-fold more toxic than was the diphtheria toxin A-chain conjugate. The C-terminal, 17-kDa region, which contains the cell surface binding site, therefore potentiates immunotoxin activity 100-fold. It has not been possible to determine whether this C-terminal translocation activity was distinct from the binding activity.
Laird and Groman, J. Virol. 19: 220 (1976) mutagenized Corynebacterium with nitrosoguanidine and ultraviolet radiation and isolated several classes of mutants within the diphtheria toxin structural gene. Leppla and Laird further characterized several of the mutant proteins and found that three of them, CRM102, CRM103, and CRM107, retained full enzymatic activity but had defective receptor binding.
Recombinant DNA technology has been used to improve immunotoxin efficacy at the gene level. Greenfield et al. (1984) in Proc. Natl. Acad. Sci. USA 80: 6953-6857, reported that they have cloned portions of diphtheria toxin and created a modified toxin which contains the N-terminal hydrophobic region of diphtheria toxin but lacks the C-terminal cysteine for ease of linking to antibodies. This fragment lacks the cell surface-binding sits of diphtheria toxin but includes most of the hydrophobic region thought to facilitate membrane transport.
Although cleavage of ricin or diphtheria toxin into A and B-chains had been thought to improve the specificity of the immunotoxins produced from the A-chain, cleavage of ricin or diphtheria toxins into A and B-chains removes the portion of the molecule containing residues important for transport into the cytosol of the cell. Specific cytotoxic reagents made by coupling toxin A subunits to antibodies have low systemic toxicity but also very low tumor toxicity. More potent reagents can be made by coupling intact toxins to monoclonal antibodies, as detailed in J. Immunol. 136: 93-98 and Proc. Natl. Acad. Sci. USA 77: 5483-5486. These reagents, however, have a high systemic toxicity due to the toxin binding to normal cells, although they can have applications in vitro in bone marrow transplantation (cf. Science 222: 512-515).
It was found by Youle et al., as reported in Jour. Immunol., op. cit., that monoclonal antibody-intact diphtheria toxin conjugates reacted quite differently from the intact ricin immunotoxins. Of the four reagents examined, a monoclonal antibody against type T3 antigen linked to diphtheria toxin (UCHT1-DT) had unique properties. This reagent showed greater selectivity in its toxicity to T cells as compared to stem cells than UCHT1-ricin. UCHT1-DT was found to be 10 to 100 times more selective than any previously reported immunotoxin.
Neville et al., in U.S. Pat. Nos. 4,359,457 and 4,440,747, disclose that the receptor specificity of toxins can be altered by coupling the intact toxin to monoclonal antibodies directed to the cell surface antigen Thy 1.2. However, the only toxin specifically disclosed to be treated in this manner is ricin. The same inventors in U.S. Pat. No. 4,500,637, disclose the covalent linkage of a monoclonal antibody known as TA-1 directed against human T-cells for use in treating human donor bone marrow before the marrow is fused into a human recipient. Thus, this reagent has been found to be useful in preventing graft versus host disease.
Another method of treating ricin to increase the rate of protein synthesis inhibition is by adding excess ricin B-chain to target cells independent of the amount of ricin A-chain bound to the cell surface membrane. The ricin A-chains used in this procedure are conjugated to anti-Thy 1.1 monoclonal antibodies. This process is disclosed in Neville et al., U.S. Pat. No. 4,520,011.
Yet another method of treating graft versus host disease is disclosed in Neville et al., U.S. Pat. No. 4,520,226. In this method, monoclonal antibodies specific for T-lymphocytes in human donor bone marrow are covalently linked to separate ricin toxin, combined in a mixture to form a treatment reagent, and combined with bone marrow removed from a human donor. The bone marrow-reagent mixture is then infused into an irradiated recipient, which virtually eliminates T-lymphocyte activity.
However, none of the prior art has shown effective immunotoxins prepared from diphtheria toxin which have the desired specificity and activity.